ChemDiv’s HCA-HCS Platform Phenotypic Assays
High-content screening (HCS), also known as high-content analysis (HCA), is a method that is used in drug discovery to identify substances that alter the phenotype of a cell in a desired manner.
ChemDiv has implemented high-content-based assays for high-throughput screening and compound validation with a primary focus on disease-process ontologies and mechanisms. With our expanded capabilities in combine high-content analysis and high-throughput screening, we are moving beyond traditional target-centric biochemical assays; toward phenotypic and genomic approaches to interrogating disease targets dynamically, and identifying new ways to design and develop therapeutic molecules. Our pathway and phenotypic screening of primary and immortalized cells opens up a wealth of information not previously available.
Our High Content Assay (HCA) platform enables investigation of complex biological responses at the cellular and organismal level (e.g. C. elegans); for example, by observing and quantifying a morphological change; providing opportunities for new insights and the ability to determine the mode of action of an active compound, even when the target is not completely known or characterized.
ChemDiv’s HCAs also permit investigation of the compound’s action on a larger number of known targets, along with detection of the compound’s side effects in order to continue only development selectively, i.e, only highly druggable lead compounds – those with a high probability being successfully developed into a drug. The use of alternative model systems such as plant germinal cells and other model organisms in conjunction with disease-relevant endpoints typifies the R&D process of innovative drugs offered by ChemDiv in the areas of human and animal healthcare and agro-biotechnology.
Table 1. ChemDiv HCA-HCS Platform Phenotypic Assays
» Anthranilic acid glucosyl esters (gut granule) deposition (C. elegans)
» Cell cycle analysis
» Differential cell death – toxicity-induced necrosis versus programmatic apoptosis
» Differentiation markers
» Endo- and phagocytosis
» Intracellular transport and aggregation
» Mitochondrial physiology and processing (distribution and mass)
» Motility (C. elegans)
» Neurite outgrowth
» Neurodegeneration and neuroprotection
» Pollen tubule outgrowth (N. tabacum L)
» Post-translational processing and modification
» Cellular proliferation
» Cytoskeletal dynamics and reorganization
» Synaptic density and structural plasticity
ChemDiv ´s high-content screening (“CD-HCS”) proficiency is based on several instrument platforms, including the ImageXpress Micro Widefield High-Content Screening system (Molecular Devices) which has all the capabilities of the standard ImageXpress Micro system, plus these significant enhancements:
- One well, one image
- Fewer images for large objects
- Statistically relevant results in half the time
- Instant-on, long-lived solid state light source
- Reliable assays with <5% CV of intensities across plate
The CD-HCS meet the challenges of the high-content screening environment where time-to-result and data qualities are equally important. CD-HCS platform offers the only true end-to-end solution for HCS, from acquisition through data analysis. To generate relevant numerical data from high-content assays, Molecular Devices’ software works at every level, from extracting features from individual cells through multi-parametric analysis and visualization of multiple assays. A scalable parallel processing solution to drive application module analysis time to be faster than acquisition. MetaXpress PowerCore Software harnesses the power of parallel processing to allow HCS users to review data as soon as image acquisition is over. Image analysis for all application modules and custom modules is sped up without compromising quality and is fully scalable, allowing HCS users to analyze screens ranging from a few plates to a million compounds. Analysis becomes up to 25-fold faster than acquisition (384-well microplate, three images per well, 2MB per image) using four complex application modules.
Streamling the CD-HCS image analytics platform enables a thoroughfare to discovery of new targets and pathways at unprecedented levels using phenotypic profiling of compounds and new Click-Chemistry based approaches to investigate compound’s mechanism of action. Our CD-HCS integrates with of extremely powerful Click-Chemistry applications to generate assays for biological problems that previously required radioactive components or techniques that were time-consuming and insufficiently robust for the rigorous of multiplexed HCS analyses. The specificity of reactions between the azide-alkyne tag pair affords detection with exceptional sensitivity and low background even in complex biological matrices.
Our Targets and Assays
Cell imaging at ChemDiv provides a large amount of information from primary screening data that can be used to distinguish compounds with differing effects on cells, and together with automated analysis, to quantify compound effects. We observed that compounds with similar target specificities and modes of inhibition cause similar cellular phenotypes (see Table 1). Based on this observation, we hypothesized that we could quantitatively classify compounds with diverse mechanisms of action using cellular phenotypes and identify compounds with unintended cellular activities within a chemical series. We are actively developing phenometrix technology as an automated system capable of quantifying, clustering, and classifying changes in cellular phenotypes for this purpose. Using this system, known compounds can be accurately classified into distinct mechanisms of action. This technology is available to ChemDiv’s clients for a variety of drug discovery applications, including high-throughput primary screening of chemical and siRNA libraries, and as a secondary assay to detect unintended activities and toxicities.Shapes not supported
Our Research Areas
HCS at ChemDiv is now a conventional building block in many drug discovery programs across a diverse research areas. Currently, CD-HCS is employed in agro-biotechnology and longevity studies, along with a number of therapeutic areas of cancer, Protein-Misfolding diseases (e.g., AD, PD, HD), diabetes, pain, neuroinflammation and neurodegeneration, and virology. Our capabilities in stem cell biology, regenerative medicine and phagotherapy are rapidly expanding as well.
Table 2. Examples of ChemDiv HCA-HCS based research
|Phenotype/Target process||Screen/model||DD project stage||Research Area|
|Calcium-Propagated Necrotic Wave/ excitotoxicity and ischemia-induced neurodegeneration||Anthranilic acid glucosyl esters (gut granules) deposition/ in vivo - C.elegans||Hit identification||Longevity|
|Motility/ spatio-temporal dynamics of organismal death.||Motility/ in vivo - C.elegans||Hit identification||Aging/ Longevity|
|Diabetic Neuropathy/ Reorganization of cytoskeleton||Tubulin & Actin rearrangement||Compound library profiling||Diabetes|
|Neurodegeneration/Amyloid & Tau oligomer binding & infectivity||Neurite outgrowth / co-culture system of primary cortical neurons||Hit identification||Protein-Misfolded diseases|
|Alzheimer’s protein aggregation||Number of aggregates/ co-culture system of primary cortical neurons||Compound library profiling||Protein-Misfolded diseases|
|Proteastasis/Autophagy||LC3 accumulation in autophagosomes/LC3-fluorophore transfected cell line||Target validation||Neurodegeneration|
|Post-translation modification/ Epigenetic control||Protein acetylation & methylation||Target validation||Neurodegeneration|
|Cell death, necrosis ,apoptosis/ B-cell lymphoma proliferation||Binding & Translocation of NBD-loaded nanoparticles/PBMC (human)||Target identification & validation||Cancer|
|Production of IL-1β & ROS/ inflammasome activation||NLRP3 inflammasome activation/ monocyte-derived macrophages (MDMs) from drug-naïve patients with newly diagnosed type 2 diabetes||Assay development||Type 2 Diabetes|
|Life span/ mitochondrial control of oxidant production, insulin signaling, and aging||Life span & lipid granule staining (relative Nile red fluorescence) / in vivo - C.elegans||Protocol development||Pain (anesthetics), diabetes, aging, PD|
|Individual pollen square/modulation of root growth||Pollen Tube outgrowth/Nicotiana tabacum L.||HTS of 10K||Agro-biotechnology|